In vitro activity: ML-161 is an allosteric inhibitor of PAR1. ML-161 displays dose-dependent inhibition of thrombin-induced platelet activation, as measured by P-selectin expression. ML-161 displays selective inhibition of SFLLRN and thrombin-induced platelet aggregation, both of which operate via PAR1, and has no effect on AYPGKF, thromboxane, or ADP-induced platelet aggregation, which are all agonists at alternative platelet GPCRs.

Kinase Assay: A high-throughput screen of the NIH-MLSMR compound collection, along with a series of secondary assays to identify potential targets of hit compounds, previously identified a 1,3-diaminobenzene scaffold that targets protease-activated receptor 1 (PAR1). We now report additional structure-activity relationship (SAR) studies that delineate the requirements for activity at PAR1 and identify plasma-stable analogues with nanomolar inhibition of PAR1-mediated platelet activation. Compound 4 was declared as a probe (ML161) with the NIH Molecular Libraries Program. This compound inhibited platelet aggregation induced by a PAR1 peptide agonist or by thrombin but not by several other platelet agonists. Initial studies suggest that ML161 is an allosteric inhibitor of PAR1. These findings may be important for the discovery of antithrombotics with an improved safety profile.